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Introduction

Bioavailability, expressed as the letter “F”, is the measurement of the effective dose of an active drug which reaches the systemic circulation from the site of administration. There are many barriers and obstacles which prevent therapeutically active drugs from reaching the target sites. These include poor adsorption from gastro-intestinal tracts, degradation or metabolism of the drug before it is adsorbed, and also the hepatic “first-pass effect.”

To measure the amount of a drug which is available in the blood after non-intravenous administration you must create a drug concentration vs. time plot for the drug after both intravenous and the non-intravenous administrations. From the plot the “absolute bioavailability” can be determined by the following formula:

F: bioavailability

AUC: area under curve

IV: intravenously

po: oral route

dose: initial dose

F: bioavailability

AUC: area under curve

IV: intravenously

po: oral route

dose: initial dose

Figure 1 – Example of a concentration vs. time plot used to calculate f values.

An important aspect to remember is that bioavailability describes the concentration of the drug which reaches systemic circulation, not necessarily the main site of action. Systemic circulation is defined as the opposite of pulmonary circulation. It is the branch of the cardiovascular system which moves oxygenated blood away from the heart and returns deoxygenated blood back to the heart. Blood is moved away from the heart in arterial systems and brought back through venous systems.

Figure 2 – Systemic flow to and from heart.

Drug Administration

There are several different ways to administer drugs. The United States Food and Drug Administration recognize 111 separate distinct routes of drug administration varying from urethral to vaginal. All of these methods of administration are focused into three broad categories: topical, enteral, and parenteral.

Figure 3- Modes of administration

Topical administration installs a local effect and is only effective when applied directly to the site of action. Examples of topical administration include topical ointments, eye and nose drops, and inhalers. Topical ointments work directly through absorption through the epidermis, eye and nose drops target the eyes and nose and are applied directly to the area, and inhalers are breathed into the lungs where they are used.

Figure 4- Intravenous, topical, and transdermal administration

Enteral administration and parenteral administration both exhibit a systemic effect. The only difference however, is their modes of entry into the systemic circulation. Enteral administrations are given through the digestive tract while the parenteral is given in some other way still maintaining the systemic effect. Examples of the enteral administration include drugs taken orally as in pill form or rectally. The best example of parenteral administration and likely the route of administration we are most familiar with are drugs given intravenously.

What Makes a Drug Bioavailable?

Lipinski’s Rule of Five

Formulated by Christopher A. Lipinski in 1997, the rule of five is a type of “rule of thumb” which is used to evaluate a compound’s druglikeness. It was based on his observation that most medically active molecules were small lipophilic compounds. It describes the molecular properties that are important for the drugs pharmacokinetics when inside the human body. The Lipinski Rule of Five states that in order for a drug to be orally active it must have:

Refinements

Studies done by Veber et al. found that what makes a drug orally bioavailable. Their results for the most part agreed with Lipinski’s rule of five; however they were able to refine the rules to an extent. Veber’s most outstanding realization was that it is not a drug’s size that makes it bioavailable, but rather the molecule’s rigidity. This was done by using a library of 1100 molecules from GlaxoSmithKline. The researchers used molecular parameters like ClogP values to determine molecules hydrophobicity, and rotatable bond counts to test for rigidity.

Descriptions of some of the molecular parameters include:

Rotatable bonds are any single bond not found in a ring structure. Also, atoms bound to terminal hydrogens are not included as a rotatable bond. This measure was used to determine a molecule’s rigidity.

Figure 6- The octanol-water partition

C logP is known as the Octanol-water partition coefficient and is commonly used to quantify a molecule’s hydrophobicity. The polar surface area (PSA) is another measure used to determinehydrophilicity/hydrophobicity. This is either done by topological PSA (TPSA) or through hydrogen bond donor/hydrogen bond acceptor counts.

Figure 7

The table below shows results from the Veber et al. experiment. It shows that regardless of molecular weight, oral bioavailability is more determined by rotatable bond count. It is easily assumed that an increase in molecular weight would make a drug less bioavailable, but in fact those compounds containing a high number of freely rotatable bonds are more rigid and therefore are orally bioavailable.

Figure 8

Absorption

Definitions

The absorption of a drug is defined as the process through which a drug travels from its site of administration to the bloodstream. Obviously, absorption is a process that does not apply to intravenous administration because the drug is injected directly into the bloodstream. Absorption typically involves the drug crossing several layers of cells as it diffuses from one tissue to another. In order to penetrate layers of cells, the drug must be capable of crossing the phospholipid bilayer membrane of cells. Absorption is the primary determinant of bioavailability, since it determines how much of the drug will reach the bloodstream. Poor absorption means that fewer drug molecules will successfully reach the bloodstream, and bioavailability will be reduced.

Figure 9- Representation of a cell membrane, including embedded proteins.

The principal component of cell membranes is the phospholipid bilayer. Although membranes vary in the amount and types of proteins embedded in them, the phospholipid bilayer is a commonality among all cell membranes. It is so named because it is composed of a polar phosphate group and a hydrophobic fatty acid tail. When two layers of this molecule associate, the non-polar hydrocarbon tails orient towards each other, while the polar and hydrophilic heads of the inner and outer layers orient themselves towards the cell cytoplasm and the external environment, respectively. This creates a membrane with a hydrophilic character on the inner and outer surfaces, and a highly hydrophobic character in between.

Figure 10- The chemical structure of a single phopholipid, and their orientation in a bilayer.

Typically, drugs that are well absorbed have some physical similarities. They tend to be small molecules (smaller than 500 Daltons), and hydrophobic as well. An uncharged, non-polar molecule will cross cell membranes more easily because they have no problem interacting with the hydrophobic interior of the lipid bilayer. This facilitates diffusion across cell membranes (as does a reduced size) and so these two characteristics are common in drugs with a high bioavailability. It should be noted that drugs lacking these characteristics may also have good bioavailability. These drugs will generally rely on transporters within the cell membrane to penetrate cells, rather than passively diffusing across them.

Ion Trapping

Ion trapping is an interesting phenomenon that occurs in drug transit between tissues or areas of different pH. Every drug has a characteristic pKa: the pH below which the drug molecule will become ionized and acquire a charge. A molecule with an acidic group will lose a proton and become negatively charged, whereas a molecule with a basic group will become protonated and positively charged. When a molecule is charged, it loses one of the key characteristics of a bioactive drug- the hydrophobicity that allows it to cross cell membranes. A charged molecule cannot cross cell membranes, and therefore cannot exit the area in which it became ionized. This leads to an accumulation of charged drug molecules in a given tissue.

Luckily, LeChateliers Principle plays an essential role in compensating for this imbalance in drug distribution. Depending on how acidic the pH of the compartment is, a certain percentage of the drug will be ionized (the farther the pH is from the pKa of the drug, the higher the proportion of ionized drug molecules will be). Because drug molecules are rarely 100% ionized, some uncharged molecules will be present. These molecules can cross cell membranes, unlike their ionized counterparts, and enter another compartment or tissue. This creates an imbalance in the ratio of ionized:unionized molecules in the original compartment. To restore the ratio, some charged molecules will lose their charge (by LeChateliers Principle) and become neutral molecules that are capable of crossing cell membranes. When they cross into the next compartment, they again induce an imbalance in the charged:uncharged ratio in the original compartment, causing more molecules to become neutral and cross the barrier into the next compartment. This cycle continues until most of the drug has moved to another compartment. This process plays an integral role in the transit of a drug from its administration site to the bloodstream, thereby influencing the drug’s bioavailability.

Figure 11- The effects of ionization on two common drugs: aspirin and morphine.

An Example of Absorption: The GI Tract

Oral administration is the most common form of drug administration because of its convenient and painless nature. In this type of administration, the drug is ingested by mouth in a pill, capsule, or liquid form and travels through the stomach to the small intestine, where absorption usually occurs. To enter the bloodstream from the intestinal lumen, a drug is required to cross both the apical (lumen-facing) and baso-lateral faces of the epithelial cells to reach the capillary vasculature lying beneath the epithelium, where it can diffuse into the bloodstream. A few exceptions to this passive diffusion across intestinal epithelium are the drugs fluorouracil and levodopa. Fluorouracil is a chemotherapeutic agent used in cancer therapy that crosses epithelial cells by taking advantage of a pyrimidine active transport system. Levodopa, used in treatment of Parkinson’s disease, uses a phenylalanine active transporter to cross cell membranes.

Figure 12- Histology of epithelial cells in the trachea. A very similiar structure is present in the epithelial cells lining the GI tract.

Figure 13- The structure of two drugs that take advantage of active transport proteins to cross cell membranes.

There are several factors that will influence a drug’s bioavailability when orally administered, regardless of the molecule’s inherent physical properties. An example of one of these factors is intestinal motility; that is, how quickly the stomach’s contents are emptying into the small intestine. When drug absorption occurs in the small intestine, it is desirable for the drug to spend as little time as possible in the acidic stomach environment to reduce it’s chances of becoming permanently altered by the harsh conditions, as well as to promote a quicker absorption and therefore quicker onset of the drug’s effect. When a meal is eaten, the intestinal motility is slowed to promote digestion and absorption of nutrients, which is why some drugs are recommended to be taken on an empty stomach. On the other hand, some drugs are recommended to be taken with food to dampen harsh effects of the drug on the stomach wall; to promote alteration of a prodrug into its active form in the stomach; or to promote absorption through the stomach epithelium if that is the preferred site of absorption.

Particle size also plays an important role in the bioavailability of a drug when administered orally. Particle size refers to the actual size of the pill or capsule, and its non-medicinal chemical composition. In the 1970’s in New York, it was noticed that certain patients needed a much higher dose of digoxin to maintain its effects compared to other patients on the exact same dosing schedule. They tested their hypothesis on 4 groups of people taking the same dose of digoxin but in four different preparations. The results were astounding: some preparations were absorbed completely and persisted in the bloodstream for several hours, while others were hardly absorbed at all. It is therefore important to take into consideration the preparation of orally administered drugs and how it may affect their bioavailability.

Figure 14 – The levels of digoxin in systemic circulation for four different preparations of the drug, containing the same amount of digoxin.

Another factor influencing the absorption of drugs in the GI tract is the chemical interactions that a drug may experience with the stomach contents. Certain foods can interfere and lower absorption in the GI tract such as dairy products. The calcium ions found in dairy can bind to a drug (i.e.-tetracycline), and prevent it from being absorbed across the cell membranes due to the positive charge covering the drug molecule. It is also important to bear in mind possible drug-drug interactions, such as the drugs colestyramine and warfarin- which bind together in the stomach to form a complex too large for either drug to be absorbed. These external factors prove that absorption and bioavailability depend on more than just the chemical properties and structure of the drug molecule itself.

Distribution

Definition

By definition, distribution is the process by which a drug travels to its target site from the time it enters the bloodstream. At first glance, this process appears to be beyond the scope of bioavailability since the drug is already in the bloodstream. However, there are obstacles that can be encountered after entering the bloodstream that can dramatically impact the amount of a drug that reaches it target site. Whether this falls within the definition of bioavailability or not, these are factors that must be taken into consideration when looking at drug efficacy.

Blood Proteins

Our blood is full of proteins whose jobs are to nonspecifically carry molecules through our bloodstream. This unfortunately means that many of these large, promiscuous proteins will sequester drug molecules as well because they have very little ability to distinguish a drug from substrates they are meant to carry. These blood transporters can include serum albumin, β-globulin, and acid glycoproteins. Also capable of trapping drugs are fatty tissues, because a highly hydrophobic drug will diffuse into this region and never diffuse out. Both fatty tissues and protein transporters prevent drugs from reaching their target site. Transporters do so by holding the drug in a complex so large that it cannot leave the capillaries; and fatty tissues do this by holding the drug in a poorly vascularized tissue (fat receives about 2% of our cardiac output) for which the drug has a very high affinity.

Blood-Brain Barrier

Another major obstacle encountered during distribution that must be considered is the blood-brain barrier. This change in vascular histology presents an issue when targeting drugs for the brain. Regardless of the levels of the drug in systemic circulation, a drug can only enter into brain tissue if it can cross this barrier. The blood-brain barrier is characterized by tight junctions between endothelial cells of the capillaries, making it unusually difficult for a drug to exit a capillary and enter the surrounding tissue. The evolutionary significance of this barrier is to protect the brain from blood-borne infections and chemicals, which unfortunately translates into a protection system against drugs as well. If a drug’s desired target is the brain, the molecule must be highly hydrophobic to ensure penetration of the capillary wall. It is worth noting that this barrier is the reason why brain infections, although rare, are often very serious: because antibodies are unable to cross the blood-brain barrier and humans therefore have no adaptive defense against infections in this tissue.

Figure 16- The histology of the blood-brain barrier, showing the diffusion of only select molecules into the brain tissue.

Metabolism

First-Pass Metabolism

Recall that bioavailability is defined once the drug reaches the systemic circulation, which requires it to be carried to the heart via deoxygenated blood in the venous system, and oxygenated in the lungs before entering systemic circulation. We generally think of metabolism as occurring after the drug has been distributed through the body via the systemic circulation, and before being excreted. However, a different kind of metabolism occurs when a drug is orally administered called first pass metabolism. When stomach contents (including an ingested drug) diffuse into the blood to be carried to the heart, they must first travel to the liver via the hepatic portal vein. This vein and the associated capillaries are the only vasculature that carry blood away from the stomach- and it leads directly to the liver, not the heart. Therefore, the path of stomach contents that have diffused into the blood is: hepatic portal vein> liver> hepatic vein> inferior vena cava> oxygenation in lungs> systemic circulation. Subjecting a drug to the metabolic enzymes of the liver may alter it to a point that it is no longer active before it even reaches systemic circulation. First pass metabolism is therefore a very important process to consider when evaluating the bioavailability of a drug. The bioavailability will decrease if a portion of the drug is rendered inactive by this process. The exception to this is prodrugs, which take advantage of first pass metabolism by being converted into an active form during its time in the liver, instead of being inactivated.

Figure 18- The hepatic portal system. The yellow vasculature is the vein system (hepatic portal vein) which carries blood away from the stomach (and other gastrointestinal organs) and to the liver before being taken to the heart.

An Example of First-Pass Metabolism: Alcohol

An excellent example of a drug that is metabolized by first pass metabolism is alcohol. Our bodies are capable of altering alcohol to an inactive form at a certain rate, which varies between individuals. The enzyme responsible for this alteration is called alcohol dehydrogenase and it is found in the liver. Its substrates are a variety of alcohols, which are oxidized by the enzyme with the help of a cofactor, NAD+, which is reduced during the process. NAD+ is also the limiting reagent of the reaction, so how quickly we metabolize alcohol depends on the amount of NAD+ in our livers. If an individual consumes alcohol more quickly than they can metabolize it, the unaltered drug is able to reach systemic circulation and produce its effects.

The reaction proceeds as follows:

1) A binary complex is formed by NAD+ binding to the ADH enzyme (not depicted in figure), forming a binding site for the substrate.

2) Binary complex formation induces (possibly) a change in the pKa of the water molecule attached to Zn, causing it to lose a proton.

3) The resulting partial + charge on Zn attracts the partial – charge of the OH group on the alcohol molecule

4) A hydrogen bond is formed between the alcohol and NAD+, creating a ternary complex.

5) The alcohol protonates the ADH enzyme with its acidic proton, reforming the water moiety on the enzyme.

6) Formation of a stabilizing double bond causes NAD and the associated hydrogen to leave as the reduced cofactor, NADH.

7) Acetaldehyde is released from the enzyme and is further metabolized in the liver by acetaldehyde dehydrogenase to eliminate the toxic molecule.

Figure 19 – The 3D structure of the active site of alcohol dehydrogenase (left); and the main reaction scheme for the oxidation of alcohol by ADH (right).

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DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 30 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international,
etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules
and implementation them on commercial scale over a 30 year tenure till date Dec 2017, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 50 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 19 lakh plus views on New Drug Approvals Blog in 216 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc